Exposure to fluoride can induce low sperm quality; however, little is known about the molecular mechanisms by which fluoride exerts its toxic effects. This study was conducted to evaluate ultrastructure, oxidative stress, and apoptosis in sperm of mice treated with 150 mg/l NaF for 49 days. Furthermore, microarray analysis was also utilized to characterize the effects of fluoride in gene expression profiling on mice sperm. An increased ROS and a decreased TAC accompanied with distinct morphological changes and significant apoptosis were observed in mice sperm from the fluoride group. Fluoride exposure also significantly elevated the protein expressions of cytochrome c and active caspase-3. In global gene expression profiling, 34 up-regulated and 63 down-regulated genes, which are involved in several sperm biological processes including signal transduction, oxidative stress, apoptosis, electron transport, glycolysis, chemotaxis, spermatogenesis, and sperm capacitation, were significantly differentially expressed. Based on these findings, it was proposed that oxidative stress induced by excessive ROS may trigger sperm apoptosis through mitochondrial impairment, resulting in decreased fertility in mice exposed to fluoride. Microarray analysis also provided several important biological clues for further investigating fluoride-induced damage in sperm morphology and functions.